biostudies-arrayexpressPhilippe DessenHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-TABM-133The aim of the experiment was to characterize by a pan-genomic approach genes which are continuously up and down regulated during polyploidization during megakaryocyte differentiation.biostudies-arrayexpressHybridization - Agilent Hybridization Protocol (Chamber type: Agilent SureHyb Chamber; Quantity of labelled extract: 1 ug; duration: 17 hours; volume: 650 ul ; Temperature in C: 60). First experiment : Dye swap of 4 samples (2N, 4N, 8N and 16N versus a pool of the four samples, all in dye swap). Second experiment : Direct hybridizations (in dye-swap) of the 4n/2n, 8n/2n et 16n/2n with 2 ug per hybridization. Add the following components in clean 1.5 ml tubes: 1 ug linearly amplified cRNA labeled with cy5 for each tumor, and 1 ug linearly amplified cRNA labeled with Cy3 for the reference (pool of tumors). Conversely, for dye swapped arrays, mix 1 ug linearly amplified cRNA labelled with cy3 for each tumor, and 1 ug linearly amplified cRNA labeled with Cy5 for the reference (pool of tumorsGrowth Protocol - In vitro growth of megakaryocytes from CD34+ cells : Cytapheresis samples from healthy individuals were obtained with their informed consent. CD34+ cells were isolated using the immunomagnetic beads technique (Miltenyi, Biotec, France) [Choi, 1995 #7]. CD34+ cells were grown for 8 days in Iscove's modified Dulbecco's medium (Gibco BRL) containing penicillin (100 U/ml), streptomycine (100 mg/ml), glutamine (2 mM) (Sigma), a-monothioglycerol (76 nM, Sigma), 1.5% deionized bovine serum albumin (BSA; Cohn fraction V; Sigma), 1/100e of Insulin-Transferrin-Selenium X (Gibco BRL), and sonicated lipids (20 mg/ml) [Debili, 1993 #12]. The culture medium was supplemented with PEG-rhuMGDF (10 ng/ml) (a generous gift from Kirin Brewery, Tokyo, Japan). Cell sorting of megakaryocytes Labeling - (500 ng, Amplification=RNA polymerases). Agilent oligo Cy5 or Cy3 probes labeling protocol. Kit used for probe labeling: Agilent Fluorescent Low input Linear Amplification kit (G2554A) adapted for small amount of total RNA (500 ng total RNA per reaction). In 1.5 ml tubes add 500 ng total RNA from sample. Add 1.2 ul T7 promoter primer and add nuclease free water (Invitrogen ref:10977-015) to bring the volume to 10 ul. Denature by incubating at 65 C for 10 minutes. Place the reactions on ice and incubate 5 min. Spin briefly. Prepare a Reverse Transcription master mix, adding for one reaction 4 ul First strand Buffer 5X, 2 ul DTT 0.1M, 1 ul dNTP 10 mM mix, 0.5 ul Random Hexamer, 1 ul MMLV Reverse Transcriptase (200 U/ul), 0.5 ul RNAse out (40 U/ul), 1 ul nuclease free water. MasterNucleic Acid Extraction - Frozen pelleted cell samples (2N, 4N, 8N and 16N) were lysed using 350 ul RLT lysis buffer (Qiagen) reconstituted with 1% beta-mercapto-ethanol immediately before use. After vortexing for 5 minutes, Total RNA was isolated using Rneasy microkit from Qiagen following the DNAse treatment and purification manufacturer's protocol. RNA-concentrations were adjusted precisely at 100 ng/microliter and measured with Nanodrop spectrophotometer (www.nanodrop.com). Quality of RNA-preparations was assessed using Lab-on-a-chip Bioanalyser 2000 technology (Agilent technologies), based on the 28S/18S ribosomal RNAs ratio. All samples included in this study displayed a ratio of ribosomal RNAs superior to 1.5.MIAME ScoreRaw DataOrganizationAssays and DataMAGE-TAB FilesArray DesignsFeature Extraction - Scanning was performed with a Agilent 2565 AA DNA Microarray scanner using defaults parameters (100 PMT, 10 um resolution, at 20 C in low ozone concentration environment. Microarray images were analysed by using Feature Extraction software version A.7.5.1 from Agilent technologies. Defaults settings were usedUnknownTranscriptomicsGenomicsProteomicsPolyploidization is a part of the normal developmental process leading to platelet production during megakaryocyte (MK) differentiation. Ploidization is mainly involved in cell enlargement, but it is not clear whether gene expression is modified during MK ploidization. In this study, human MKs were grown from CD34(+) cells in the presence of thrombopoietin and sorted according to their ploidy level. A pangenomic microarray technique was applied to compare gene expression in 2N-, 4N-, 8N-, and 16N-sorted MKs. Using hierarchical clustering, we demonstrated that 2N and 4N MKs or 8N and 16N MKs are 2 different close populations with 105 discriminating genes. In the second approach, we determined the profile of genes that were continuously down- and up-regulated during polyploidization. Among ttranscription profiling by arrayHomo sapiensInterrelation between polyploidization and megakaryocyte differentiation: a gene profiling approachPhilippe DessenRaslova, Hana; Kauffmann, Audrey; Sekkai, Dalila; Ripoche, Hugues; Larbret, Frederic; Robert, Thomas; Le Roux, Diana Tronik; Kroemer, Guido; Debili, Najet; Dessen, Philippe; Lazar, Vladimir; Vainchenker, WilliamfalseTranscription profiling of human tetraploid and polyploid (8N and 16N) megakaryocytes to identify genes which are up and down regulated during megakaryocyte differentiation/polyploidizationThe aim of the experiment was to characterize by a pan-genomic approach genes which are continuously up and down regulated during polyploidization during megakaryocyte differentiation.2007-04-16T00:00:00Z2021-09-27T18:25:53Z2021-09-27T18:25:53ZE-TABM-13317170127EFO_000276817170127